Cellular Localization Of Nonsense MRNA Recognition And Decay Factors In Euplotes Octocarinatus

In eukaryotic cells,m RNA decay is one of important process of gene expression,which determines the abundance of m RNA in cytoplasm.Nonsense-mediated m RNA decay(NMD)is translation-coupled m RNA decay mechanism.It can distinguish and decay abnormal transcripts containing premature termination codons(PTCs)to avoid the expression of potentially toxic truncated proteins.NMD pathway can effectively surveil the quality of m RNA and ensure the accurate expression of gene.Although the study of NMD pathway has made some progress in mammalian,Drosophila and yeast metazoan cells,the mechanism of recognition and degradation of nonsense m RNA has not been clearly explained,and there are still many controversies.Protozoa,as a kind of single-celled eukaryotes,have some preliminary studies on the mechanism of NMD pathway,but the related research reports are relatively scarce compared with metazoan.Due to its evolutionary particularity and simplicity of cell structure,the study of protozoan NMD pathway is helpful to elucidate the evolution and molecular mechanism of eukaryotic NMD pathway.Based on the bioinformatics analysis of the key factors in the process of recognition and decay of nonsense m RNA in Euplotes octocarinatus cells,the co-location analysis of these key factors in the cells was carried out by using the Macronuclear artificial chromosome containing fluorescent protein gene.The main results are as follows:1.Based on homologous sequence search,11 NMD pathway related factors were identified from the Macronuclear genome database of the Euplotes octocarinatus.It was summarized as follows: nonsense m RNA recognition factor poly(A)binding protein(PABP);NMD triggering protein factors Up-frameshift 1(UPF1)and Up-frameshift 2(UPF2);component protein factors of exon-exon junction complex(EJC)– MAGO(Mago nashi),Y14(Tsunagi or RMB8),e IF4AIII(Eukaryotic initiation factor 4A3),and UAP56(U2AF56 Associated Protein 56);protein factors involved in decay of nonsense m RNA--exosome and processing body(P-body)protein factors DCP2(Decapping m RNA 2),XRN1(5?-3? exoribonuclease 1)and POP2(PGK promoter directed over production).2.The functional domains of each factor were predicted by Inter Pro Protein to further understand the function.3.The Swiss Model and Zhang Lab(I-TASSER)were used to predict the tertiary structure of UPF1 and UPF2 proteins online,and the ZDOCK SERVER was used to dock the two molecular structures online.After docking,PDBe PISA was used to analyze the key amino acid sites of interaction.In these two protein interactions,the key amino acids predicted to play a key role in the UPF1 protein are Val108,Val111,Phe145,Leu147,Ile186,and Lys189.The key amino acids in the UPF2 protein are Phe772,Leu776,Met779,Ile780,Phe784,and Lys786.4.The gene corresponding to the ?A-helix structure in the UPF2-U1 BD domain was deleted by site-directed mutagenesis,then by the Pull-down experiment,we confirmed that UPF2 interacts with UPF1 through its conserved ?A-helix structure in the Euplotes octocarinatus,which is the same as the molecular mechanism of the interaction between the two proteins in human cells.5.In the cell,these factors of the NMD pathway were located and colocated by the Macronuclear artificial chromosome of Euplotes octocarinatus.By using fluorescence co-localization approach,we confirmed the interaction relationship between UPF1 and UPF2,among protein factors of EJC components,and among protein factors of P-body components,respectively.Then,the EJC-dependent NMD model was confirmed by fluorescence colocalization of UPF2 with MAGO and Y14,respectively.According to the fluorescence co-localization results of UPF1 with DCP2 and exosome,respectively,we inferred the degradation of nonsense m RNA via two modes: one was that nonsense m RNA was mediated into P-body,and then 5?-Cap and 3?-Poly(A)tail was removed by DCP2 and POP2,respectively.The other was that marked nonsense m RNA directly recruited POP2 and deadenylation commences in the cytoplasm.After that,the nonsense m RNA was decayed by exosome in 3? to 5? direction.Main conclusions of the study: According to the results of this study and the previous research results of our group,the NMD pathway of Euplotes octocarinatus was speculated.(1)In terms of the recognition mechanism of nonsense m RNA,the core factors UPF1 and UPF2 may be recruited in an EJC-dependent manner to combine with the eukaryotic releasing factor and EJC to form the "URF" complex,and the interaction between UPF2 and UPF1 may be through its conservative ?A-helix domain.(2)In terms of the degradation mechanism of nonsense m RNA,nonsense m RNA may be mediated into the P-body and then decayed in 5? to 3? direction by XRN1 after being capped and deadenylation by DCP2 and POP2.Or the nonsense m RNA was directly decayed in the cytoplasm in the direction of 3? to 5? under the action of exosome after POP2 deadenylation.